Cumulative observations by our group led us to hypothesize that the molecular processes by which p53 and p63 regulate urothelial differentiation are causally linked to their role in bladder tumor initiation and progression. Our main objective is to define the critical functions of p53 and p63-isoforms in urothelial development, bladder cancer initiation and progression. For this purpose, we will use state-of -the- art molecular pathology and genetic approaches utilizing in vitro cell-based assays and in vivo mouse models, validating significant findings in human normal and tumor bladder tissues. The specific aims are: Aim #1. Comprehensive analysis of p53 and p63-isoforms during urothelial development. We discovered that p63 is essential for urothelial differentiation, and that p63-null mice developed an abnormal mono-cellular layer urothelium. Preliminary data supports that this layer is constituted of umbrella cells. This aim has two subaims: 1A) To further define the p63 dependent pathway of urothelial differentiation (basal & suprabasal layers) vs. p63 Independent pathway (umbrella cells). We will define gene expression profiles of microdissected cell types of murine and human urothelium. p53 and p63 functional status will be assessed in distinct urothelial cells, and in recently identified candidate urothelial progrenitor/stem cell. 1B) To model urothelial development in the mouse using RNAi models. We are generating transgenic mice targeting TA and DeltaN p63-isoforms using a novel shRNA conditional approach. This will allow analysis of how suppression of p63 variants affects urothelial development, and their impact on the adult urothelium. Aim #2. Molecular and functional studies of p53 and p63-isoforms in bladder cancer progression. We will correlate TP53 mutational status and p63-isoform expression in non-invasive and invasive bladder carcinomas, deciphering the biological activities of these p53-family members in bladder cancer. Recent data from our laboratory reveals that mutant p53 and p63 DeltaN display negative cooperative effects, and that invasive bladder cancer could be categorized into two subgroups based on p63-isoforms expression. This phenomenon will be mechanistically explored using in vitro approaches. Aim #3. Role of p53 and p63-isoforms in bladder cancer stem cells and the resistance phenotype. We plan to further characterize a candidate bladder cancer stem cell that we recently identified using gene/protein expression analyses. Our studies will center on defining the functional significance of p53 and p63-isoforms in bladder cancer stem cell homeostasis. We will evaluate the clinical relevance of the identified cancer stem cell by performing quantitative analysis of its cell load as it relates to chemoresistance, and by testing novel therapeutic compounds targeting this cancer stem cell population.